ES2549455T3 - Method for urinary detection of bladder cancer - Google Patents

Abstract

A method for in vitro detection of bladder cancer in a patient, characterized in that it comprises the steps of: (i) extracting the DNA contained in a urine sample taken from said patient; (ii) fragment the DNA extracted in step (i); (iii) labeling the DNA fragments obtained uniformly with a labeling agent so that a set of labeled DNA is formed; (iv) forming at least one aliquot of the set of labeled DNA and contacting each aliquot with a set of reference DNA, said contact being carried out under conditions that allow specific hybridization of the DNA fragments labeled with said reference DNA ; said reference DNA comprising the DNA sequences included in each of the following loci present in human chromosomes: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7 , 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22; (v) remove labeled DNA fragments that do not specifically hybridize with the reference DNAs; (vi) determine the intensity of the signal produced by the hybridized labeled fragments with each of the selected reference DNAs; (vii) determine, for each reference DNA, the deviations between the signals obtained in comparison to those obtained with a control DNA of a healthy patient; (viii) derive the patient's cancer stage from said deviations observed for loci 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q , 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22- qter, 17p, 19 and 22.

Description

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DESCRIPTION

Method for urinary detection of bladder cancer

The invention relates to the diagnosis of bladder cancer and more particularly to the urinary detection of the transitional type of bladder carcinoma (CBT: transitional cell carcinoma).

Transitional type carcinomas are found in transitional epithelial tumors, which comprise approximately 70% to 90% of epithelial bladder tumors. They are characterized by their great morphological variability, so that their prognosis is sometimes difficult to predict.

In terms of histology, urothelial tumors can be papillary or non-papillary, of high or low malignancy (histological grade), infiltrating or non-infiltrating, with each lesion being the combined results of these three morphological characteristics.

There are several classifications for urothelial bladder tumors. The classification most commonly used so far has been that of WHO, which has existed since 1974. It classified transitional cancers of the epithelium as benign (exophytic papilloma, inverted papilloma) or malignant (transitional epithelial carcinomas grade 1, 2 and 3). In 1998, after several meetings involving anatomical pathologists, urologists, oncologists and biologists, a consensus classification of urothelial tumors was established and validated. It is this new classification (Epstein J.I., Am. J. Surg. Pathol. (1998), 22: 1435-1448) that is currently used. Although it is only slightly different from the previous WHO classification, it has the advantage of greater concordance with the biology and evolutionary potential of urothelial tumors.

According to this classification, bladder tumors are classified by "grade", from G1 to G3, according to different cytomorphological criteria. The higher the grade, the less differentiated the appearance of the cells and the more aggressive they are.

Tumors are also classified according to their superficial characteristics (pTa and T1) or invasion capacity in the muscle (from T2 to T4). Low-grade tumors are generally non-invasive or have infiltrated the superficial layers (stages Ta and T1), while high-grade tumors are more aggressive and are often detected in T1 or a more advanced stage.

The determination of the degree of tumor cells is of critical importance to the doctor, as it will help to decide which therapeutic methods should be used.

While the presence of invasive grade 3 tumors requires total ablation of the bladder and its auxiliary glands, most of the low-grade / stage tumors (Ta and T1) can usually be treated locally while remaining intact, through different therapeutic strategies. such as chemotherapy, immunotherapy or BCG therapy.

Patients suffering from a low-grade, non-invasive tumor generally have a good prognosis, but they have to undergo periodic monitoring since the risk of cancer relapse is of the order of 70%. Consequently, patients should be monitored on a regular basis after treatment, every three months during the first two years, and every six months thereafter, and in the case of a relapse it is very important to monitor the progression of tumors.

In addition, low-grade Ta tumors invade the muscle only in 10-15% of cases, while T1 tumors progress to T2 grade in 30-35% of cases. High-grade tumors progress more rapidly, patients rapidly reaching grade T2, and remote metastases often form over the course of the next two years.

Currently, only invasive clinical examinations, in other words that involve biopsy, direct endoscopic visualization of tumors, or surgery, can make it possible to establish a reliable diagnosis of the degree of tumor cells, and the implementation of appropriate therapy.

Even so, sometimes endoscopy is unreliable and difficult to interpret. Regarding cytology after a biopsy, several studies show that its reliability is mediocre, resulting in the failed detection of 50% of tumors (Boman, H., et al., 2002, J. Urol., 167 ( 1): 80-83).

Due to the high prevalence of bladder cancer in the population, particularly in people over 50, and with the aim of improving both the diagnosis and the comfort of patients, considerable research efforts have been devoted to the development of a detection Non-invasive indirect bladder cancer.

In this regard, several research teams have attempted to detect urological tumors using urinary samples using conventional tumor marker proteins.

However, until now, it has been found that the detection of these proteins is not specific enough to be reliable with respect to the origin of the proteins and the stage of tumor development.

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In 1999, an American team described the possibility of following bladder cancer progression by analyzing microsatellite DNA sequences contained in the urine (Steiner et al. (1997) Nature Medicine 3: 621-624). This detection method, which consists in amplifying and measuring the variability of some repeated non-coding mitochondrial DNA sequences, is based on the observation that these sequences are generally altered in the carcinoma cells.

However, this technique is not completely satisfactory, while it does not effectively determine the grade of the tumors.

Other molecular methods are based on PCR methods, which attempt to amplify or detect genetic markers, such as local mutations in tumor suppressor genes, applied to the DNA present in the patients' urine. However, these methods require specific primers comprising 20 to 40 nucleotides, which are not easy to develop, in view of the size and variability of the human genome. In addition, as soon as the DNA of the tumor cells is mixed in the urine with the DNA of the healthy cells, the desired amplification is masked with that caused by the latter.

Comparative genomic hybridization based on microarray (CGH in microarray) is a method that has been used to identify genetic markers associated with certain pathologies. Snijders et al [Snijders et al (2001) "Assembly of microarrays for genome-wide measurement of DNA copy number" Nature Genetics, (29)] describe the HumArray 1.14 matrix containing 2,460 BAC and PI clones that encompass the human genome.

The microarray-based comparative genomic hybridization strategies were first used to identify some potential markers of bladder cancer in 2003 [Veltman et al. (2003) "Array-based Comparative Genomic Hybridization for Genome-Wide Screening of DNA Copy Number in Bladder Tumors" Cancer Research, 63, 2872-2880]. However, in this study no statistically significant association was observed between the pattern of alterations in the number of copies and the stage or grade of the tumor.

As a result of the difficulty of using the different known genetic markers of the transitional type of bladder carcinoma, the inventors prefer the use of an innovative strategy based on the simultaneous detection of several genetic abnormalities frequently found in CBT cells.

For this purpose, they listed the main genetic abnormalities found in transitional bladder carcinomas and selected the most representative of the grade of tumor cells. In this strategy, they gave preference to genetic abnormalities that consist of physical alterations in chromosomes, such as ploidies or deletions, which typically result from the karyotype analysis of tumor cells. Then, they developed an innovative procedure that allows the simultaneous detection of the presence or absence of selected genetic abnormalities in the DNA contained in the urine of the patients.

More particularly, the inventors have selected a specific matrix of 25 human chromosome loci (using matrix-based chromosome hybridization comparative techniques with respect to the total DNA extracted from a urine sample) providing a significant fluorescent signal.

Until now, trials based on urine samples that offer the same levels of specificity and sensitivity, and that are also able to clearly distinguish between low and high grade tumors have not been available (Budman LI et al., 2008 CUAJ 2 ( 3): 212-221).

This method has an incredible ability to diagnose the presence of transitional bladder carcinoma cells, without the need for surgical examination of the bladder, while providing a good indication of the extent of these tumor cells.

Fig. 1: Classification of the tumors analyzed in the examples, according to the usual clinical criteria.

Fig. 2: classification of patients whose urine was analyzed in the examples, as a function of the results obtained using endoscopy and cytoscopy.

Detailed description of the invention

The object of the present application is to provide a method for the detection of bladder cancer, more particularly the urinary detection of bladder cancer.

This detection is obtained through the labeling of the total amount of DNA in a patient's urine, and the detection in this DNA of chromosomal sequences comprised in loci, which could potentially be affected by genetic abnormalities.

These chromosomal sequences, which are the reference DNAs according to the present invention, could potentially be absent in the case of deletion of the chromosomal locus to which they belong, or duplicated, such as in the case of a trisomy.

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Tumor cells in bladder cancer are often the source of chromosomal reorganizations, which is indicative to some extent of the tumor character of the cells. The regions affected by this chromosomal reorganization are however potentially numerous and of varying degree. Indeed, although some loci, in particular those that encode tumor suppressor genes or are involved in cell cycle regulation, are more systematically altered, chromosomal abnormalities arise when intra and inter-chromosomal recombinations occur between homologous regions, which do not They are always the same.

According to a first embodiment, an objective of the present invention is to provide a method for in vitro detection of bladder cancer in a patient, characterized in that it comprises one or more of the following steps:

In a first step (A), the DNA contained in a urine sample taken from said patient is extracted using one of the numerous methods known to one skilled in the art. As mentioned above, the present invention advantageously allows to determine the cancerous condition of the bladder based on the urine of the patients. This method must therefore concentrate the DNA contained in the urine sample, DNA that is in its residual state since it derives, in part, from cells present in the bladder. Since DNA is not a highly fragile material, it is not necessary to take drastic measures in order to preserve the urine samples to be analyzed. Samples can be taken in the patients' house. The extracted DNA is complete, in the sense that it contains the total recoverable DNA present in the urine, whether or not it originates from the tumor cells.

In a next step (B), the DNA extracted in step (i) is fragmented, preferably by sonication, into DNA fragments with a size mostly greater than 500 base pairs (bp), preferably greater than 800 bp , and more preferably between 1,000 and 5,000 bp. Alternatively, the DNA can be digested by enzymes such as restriction enzymes.

In a next step (C), the DNA fragments obtained are uniformly labeled, by a first marker agent so that a set of labeled DNA is formed. Thus, the total DNA extracted from the urine is marked. By "uniformly" it is meant that the labeling occurs in a non-specific manner so that two different DNA fragments with an identical size are marked with the same intensity. To obtain such labeling, nucleotides coupled to a fluorophore are preferably used, which are introduced into the DNA during the course of a replication step. This step of in vitro enzymatic replication can advantageously increase the number of copies of the labeled DNA fragments available in the DNA set. The set of labeled DNA is preferably comprised of fragments that have random cuts.

In a next step (D), at least one aliquot is formed from the set of labeled DNA, said aliquot being able to form, depending on the circumstances, the set of total labeled DNA. Each aliquot is contacted with one, and preferably several, reference DNA, each of these reference DNA corresponding to a different DNA sequence included in the locus of a chromosome that is likely to be affected by a genetic abnormality. Said genetic anomaly is preferably correlated with a specific stage of development of bladder cancer and more particularly with a stage such as that mentioned above. The reference DNA may have been chosen, for example, according to the data taken from the scientific literature or biopsy analysis results.

According to the present invention, the reference DNAs are comprised of human chromosome DNA sequences with a length between 1,000 and 200,000 base pairs (bp), preferably a length between 2,000 and

180,000 bp, and even more preferably a length between 5,000 and 160,000 bp, such as those cloned in BAC (Bacterial Artificial Chromosome) or YAC (Artificial Yeast Chromosome) clones constructed from human genomic sequences. Such BAC clones are described in publicly accessible databases relevant to the human genome, such as those of CNBI or the UCSC.

Thus, while it has been proven that it is difficult to diagnose bladder cancer based on isolated markers, in particular markers corresponding to short genetic sequences that allow genetic amplification assays (PCR, TMA, etc.) to be carried out, the detection method according to the present invention it is based on a detection method that uses DNA sequences that are generally greater than 600 bp.

The inventors have established that it is preferable to use reference DNA whose sequence is included in the human chromosome loci chosen from: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22 as referred to in Table 1. The method according to the invention involves DNA of reference comprising the DNA sequences included in each of the following loci present in human chromosomes: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22.

According to the present invention, the contact between the labeled DNA fragments and the reference DNAs is carried out under conditions that allow specific hybridization, and preferably under astringent conditions such as those described in the experimental part of the present application.

In parallel to stage (D), a stage (E) consists of preparing a set of control DNA taken from one or several individuals who do not suffer from bladder cancer. Ideally, this DNA is derived from urinary samples taken from several individuals who do not suffer from cancer, randomly chosen from the human population. It is also possible to use DNA

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of human reference prepared commercially. As in the case of DNA extracted from the patient's urine, the control DNA is uniformly labeled by a second marker, preferably fluorescent. This marker is preferably different from that used to label the patient's DNA.

As in step (D), an aliquot of the labeled control DNA is contacted with the selected reference DNAs, which are identical to those used in step (D). This contact operation can be carried out separately or together, under the same hybridization conditions to those used in step (D). When the contact operation is carried out jointly, this implies that the patient's DNA and the control DNA are contacted, preferably simultaneously, with the same reference DNA.

In a next step (F), the labeled DNA fragments, which did not specifically hybridize with the reference DNA in steps (D) and (E), are removed. This step of washing non-hybridized DNA is carried out according to the procedures known to those skilled in the art, without any particular difficulty.

In a next step (G), the intensity of the signal produced by the labeled fragments that have hybridized with each of the selected reference DNAs is determined. The type and intensity of the signal depends on the type of marker used. When the first and second markers used have equivalent intensities, such as for example in the case of cyanines 3 and 5, the observed variations between the patient's DNA and the control DNA are directly related to the amount of hybridized DNA fragments located in Each reference DNA.

In a next step (H), the deviation between the recorded signals of the patient's DNA and the control DNA for each reference DNA is determined.

According to a preferred embodiment of the present invention, the DNA fragments taken from the patient in the stage

(C) They are divided into two sets, marking one using a first marker and marking the second using a second marker. The control DNA fragments of step (D) are also divided into two sets, one being marked using the first marker and the second using the second marker. With this procedure, a cross determination of the signal deviation can be determined with the first and then with the second marker. If the observed deviations between the control DNA and the patient's DNA are of the same order, regardless of the marker used, this is indicative of significant deviations.

In a next stage (I), the cancerous stage of the patient is obtained from the deviations observed in the previous stage. Thus, according to the present invention, the greater the number of deviations obtained, the more accurate is the detection of bladder cancer. In addition, the deviations provide a qualitative indication of the stage of the tumors involved, in particular when several loci that indicate the tumor grade are used simultaneously.

Alternatively, if a locus is considered to be a highly representative marker of the malignancy of the tumor cells, several reference DNAs from the same locus can be selected in order to verify the result derived from this locus.

The method according to the present invention thus allows, in step (I), to compare several reference DNAs and establish an extended table of the genetic abnormalities found. During this stage, it may be useful to weigh the results obtained for each reference DNA, in order to take into account, for example, for a tumor at a given stage, the probability of finding a genetic abnormality in the selected locus. When several reference DNAs are used to detect an anomaly in the same locus, it is also possible to weigh the deviation obtained for a reference DNA, thus taking into account its weight with respect to the other loci represented. This weighing, or use of constants in order to adjust correlations between different loci, can be used to design an algorithm that allows determining a "theoretical stage" based on the data collected during stage (H).

The above method can be summarized as a method for in vitro detection of bladder cancer in a patient, characterized in that it comprises the steps of:

(i)

 extracting the DNA contained in a urine sample taken from said patient;

(ii)

 fragment the DNA extracted in step (i);

(iii) labeling the DNA fragments obtained uniformly with a labeling agent so that a set of labeled DNA is formed;

(iv) forming at least one aliquot of the set of labeled DNA and contacting each aliquot with a set of reference DNA, said contact being carried out under conditions that allow specific hybridization of the DNA fragments labeled with said reference DNA ;

said reference DNA comprising the DNA sequences included in each of the following loci present in human chromosomes: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7 , 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22;

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(v)

 remove labeled DNA fragments that do not specifically hybridize with reference DNAs;

(saw)

 determine the intensity of the signal produced by the hybridized labeled fragments with each of the selected reference DNAs;

(vii) determine, for each reference DNA, the deviations between the signals obtained in comparison to the 5 obtained with a control DNA of a healthy patient;

(viii) derive the patient's cancer stage from said observed deviations.

The loci, which are preferably encompassed by the reference DNAs according to the present invention, are summarized in Table 1.

The reference DNAs correspond to one or several sequences included in the corresponding sequences as referenced by the NCBI (start and end positions in the genomic sequence of each chromosome).

Table 1

Location of the reference DNAs

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image 1

The characteristics of the relevant loci can be further defined as follows: 3p: A region that has several candidate genes involved in numerous forms of cancer.

4: A region more involved in tumor progress than in its beginning. In this region, 5 tumor suppressor genes have been described, targeted for other cancerous pathologies.

5p12-p13: A region involved in tumor aggressiveness, in particular metastatic spread.

6q: A region involved in high-grade tumors, which invade the muscle. The M6P / IGF2R gene is indicated as a genetic indicator in the locus 6q26-q27.

8p: A region frequently deleted in high-grade invasive tumors (especially 8p21-pter) 10 including tumor suppressor genes: EXTL3, WRN and PRLTS.

9p or 9 full: A region that is frequently affected soon. The p16 / MTS1 / CDKN2A / INK4A and p15 / MTS2 / CDKN2B genes are strong kinase inhibitors in cell cycle progression. Frequently affected

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In pTas, p16 is an anti-oncogene that depends on Rb, as will be discussed later. INK4A can act on the control points of p53 in particular.

10q or 10 complete: A minimum region 10q11-q21 and 10q24-q25: involved in many other cancers (lymphoma, prostate, colon). This area includes numerous tumor suppressor genes. The 10q deletion could be a factor that results in an incorrect prognosis.

11p: A region that corresponds to tumor aggressiveness, the first threshold in the possible direction of tetraploidy. The deletion could influence the expression of the KAI1 gene (11p11.2), which is a metastatic suppressor gene. The EXT2, WT1, TSG101 and TSSC5 genes are also involved in other cancers.

13q14: A region that corresponds to RB1 in advanced degrees and stages of cancer, RB cooperates with P53. The inactivation of RB facilitates tumor progression by regulating the onset of the S phase. Other genes may be involved, outside the locus q14, thus explaining the more extensive deletions generally detected.

14q22-qter: A region that seems to be associated with tumor progression. The deletion could be caused by unbalanced translocation. At least one tumor suppressor gene is present in this locus.

16q: A region associated with tumor progression. CDH1 encodes cadherin E, so that the deletion results in clinical and biological aggressiveness of the tumor cells. Another gene encodes cadherin H, which normally inhibits cell growth.

17: A deleted or mutated region, which occurs at a late stage, and is associated with tumor progression rather than onset. The deletion of p53 in 17p13.1 results in an increase in the number of genetic alterations due to an absence of control of the P53 protein. The HIC1 gene in 17p13.1 may also be involved.

18q12 or 18 complete: A region typically altered in tumors that invade the muscle (T2 or more). Smad4 / DPC4 and Smad2 / MADR2 / hMAD2 / JU18-1 act on RB and p15 inactivating them.

1q22-q25: An amplified region that corresponds to the transition between Ta and T1. P73 is frequently overexpressed, and thus potentially altered by duplication in this area.

3q23-q24-q25-q26-qter: A region already identified as tending to the alteration, although its implication has not been demonstrated.

Isochromosome 5p (gain of 5p and associated loss of 5q): A region in tumors that invade the muscle, altered by the activation of proto-oncogenes.

6p22: An amplified region that may be associated with a tumor invasion due to the possible presence of proto-oncogenes.

7: A region that includes the ERBB1 / EGFR gene in 7p14-p21, which encodes an EGF (epidermal growth factor) receptor, which plays a role in cell growth. In 7q31, the cmet gene encodes another growth receptor, HGF / SF (hepatocyte growth factor / dispersion factor). These areas seem to be involved in the chaotic cell growth of tumors.

8q: A region frequently involved in the Ta-T1 transition and progression to an invasive and especially metastatic tumor, through the presence of oncogenes in the 8q22-q23 region.

10q22-q23: A region that is rarely amplified, and more commonly deleted (see above).

11: Amplification of at least the 11q13 band with the CCND1 gene that controls the entry into the cell cycle. CCND1 is activated in many cancers but seems to have little correlation with cancer progression. This amplification would entail a relationship with the early stages of cancer growth.

12q15: A region that contains the MDM2 gene, which interacts with p53 in some tumors in order to suppress its role by masking its activation site.

13q33-q34: The possible trisomy 13, occasionally found in bladder tumors, could lead to duplication of this region that possibly contains a proto-oncogene.

17q: A commonly duplicated region that includes the ERBB2 gene in 17q21. This anomaly is more common in advanced degrees and stages, or is associated with relapses.

18q11: A region recognized by some rare significant amplifications.

20: A common region, most frequently restricted to the 20q locus. Proto-oncogenes of carcinoma of the familial form are present in locus 20q11-q12. In 20q13, STKIS / BTAK encodes a protein that induces chromosomal instability.

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According to a preferred aspect of the invention, the in vitro bladder cancer detection method involves testing for ploidy of the patient's urinary cell DNA based on one, several, or all of the following loci: 1q22-q24, 5p, 6q22 , 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 9p, 9q, 10q, 11p, 14q22-qter, 17p, 18q12, 19 and 22.

According to an alternative embodiment of the present invention, said method involves testing for DNA ploidy in the patient's urinary cells for one or more of the following loci: -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12 and 17p; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p and 11p; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p and 7q; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q and 1q22-24; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24 and 11q13; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13 and 8p; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p and 14q22; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22 and 22; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22 and 5p; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p and 6q22; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22 and 7;

-1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7 and 12q15; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15 and 15;

-1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15

and 6q25-q27; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15, 6q25-q27 and 19;

-1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15,

15, 6q25-q27, 19 and 13q; -1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15, 6q25-q27 , 19, 13q and 16;

Y

-1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 17p, 11p, 7q, 1q22-24, 11q13, 8p, 14q22, 22, 5p, 6q22, 7, 12q15, 15, 6q25-q27 , 19, 13q, 16 and 17q. A further aspect of the present invention involves the use of all reference DNAs taken from the 25 loci

indicated above in order to determine the grade and aggressiveness of a tumor.

Preferably, the number of different reference DNAs useful for the implementation of the method is between 50 and 500, preferably between 50 and 400. In this regard, the present invention also relates to a microarray or DNA chip, more particularly useful.

for the urinary detection of bladder cancer, characterized in that it comprises on its surface several reference DNA deposits, different from each other, each of these deposits corresponding to a sequence included in one

or more of the loci described above. This microarray or DNA chip, which is preferably a glass slide, allows the method according to the present invention to be implemented on a reduced surface area thus allowing simultaneous comparison of the haploid or polyploid state of the patient's DNA in the normal diploid state of a healthy DNA for one, several or all of the aforementioned loci.

Said DNA microarray for the detection of bladder cancer according to the invention is preferably characterized in that it comprises between 50 and 1,000 deposits of reference DNA on its surface, said reference DNA consisting of sequences included in each of the previous loci of human chromosomes. : 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q , 11p, 14q22-qter, 17p, 19 and 22.

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According to the method described previously, the DNA microarray according to the invention ideally comprises between 5 and 1,000, preferably between 5 and 400 DNA, and more preferably between 300 and 400 deposits of reference DNA as defined above on its surface. The number of deposits is reduced compared to genomic microarrays for extensive genome analysis to reduce background noise. This number is considered optimal by the inventors to improve the sensitivity of the assay with respect to the detection of bladder cancer.

By DNA chip is meant a flat glass, silicon or plastic substrate, in which known DNA nucleic sequences, such as reference DNAs according to the present invention, are placed.

Preferably, according to the present invention, the reference DNA that is placed on the DNA chip comprises the DNA sequences included only in the loci in which the chromosomal abnormalities related to bladder cancer have been described, and preferably in one, several or all of the loci described in the present application.

The present invention also relates to a kit containing one or more of the following elements:

- one or more reference DNA according to the present invention as described above, attached to a glass substrate, preferably in the form of a DNA chip or microarray;

-a DNA control sample taken from a healthy patient;

-a total DNA marker reagent as described above; Y

-a software program that allows analyzing the comparison between the diploid state of the patient's DNA and the normal diploid state of a control DNA.

Said kit is preferably packaged in a box so that all the tubes, flasks and reagents necessary to implement the method according to the present invention are accommodated.

Additional features and advantages of the invention are provided in the following non-limiting examples.

EXAMPLES

I -Preparation of the glass slide

Thanks to the availability of public databases (UCSC and NCBI), a series of BAC clones (parts of human genome DNA) were selected to encompass the chosen markers listed in Table 1. The position of these clones was confirmed by sequencing. of the two ends of the cloned DNA in order to verify the exact position of the DNA sequence in the genome. Each of these clones forms a reference DNA as implied by the present invention.

All of the selected clones encompasses all the chromosomal loci chosen as markers, making a total of 341 BAC clones.

The DNA was extracted from the BAC and amplified using repliG technology (Qiagen, Germany). Each DNA was placed so that 341 wells are formed in a PCR plate, each containing 10 μg of purified DNA. Using a Biorobotic robot and 4 needles, the DNA was placed in very small quantities on a glass slide. The design, produced by ArrayGenomics, incorporates two hybridization areas allowing, as a precaution, to carry out a twin experiment. Each DNA is randomly placed 5 times in each hybridization area in order to obtain 5 independent measurements.

The DNA is chemically fixed on a glass slide which allows it to be stored for 3 to 6 months.

II - Genetic profile of bladder tumors

Frozen tumors were collected. The chromosomal DNA of these tumors was extracted using a commercial kit. Tumors that are considered to be of a "low grade" are in the pTa / G2 stage. Tumors that are considered to be of a "high grade" are at least in the pTa / G3, pT1 and pT2 stage. Tumors are classified according to standard clinical criteria (Figure 2).

DNA extracted from frozen tumors is quantified by fluorometry (QuBit system, Invitrogen).

The human genomic DNA used as a control sample for the implementation of the diagnostic method is commercial DNA (Promega, human genomic DNA).

III -DNA marking and hybridization with reference DNAs

Complete DNA labeling

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one.

2.3 μg of control DNA is fragmented by sonication in a 55 μl eppendorf tube using a sonicator (Elmasonic sonicator) for 12 to 13 seconds.

2.

200-300 ng of the DNA prepared in this way is evaluated by electrophoresis in a 0.8% agarose gel, in order to verify that the DNA is fragmented correctly and that most of the fragments are larger than 600 pb.

3.

 The fragments are purified on micro-columns (NucleospinExtract II).

Four.

 25 μl (~ 1 μg) of aliquots of purified DNA and control DNA are placed in centrifuge tubes for labeling with Cy5 and Cy3 fluorophores.

5.

20 μl of random sequence primers (tube 1, Enzo Kit) are added to each tube, vortexed and centrifuged.

6.

 The tubes are heated in a water bath at 990C for 10 minutes.

7.

 The tubes are placed on ice for 5 minutes, centrifuged briefly and put back on ice.

8.

 5 µl of a mixture of cyanine-labeled nucleotides are added to each tube (tube 2 or 3, Enzo Kit).

9.

 In addition, 1 µl of Klenow enzyme fragments (Tube 4, Enzo Kit) is added.

10.

 The mixture is incubated in a heating block for 3 hours and 30 minutes at 370C.

eleven.

 5 μl of Stop Buffer (tube 5, Enzo Kit) are added after which the tubes are placed on ice. Washing of the labeled DNA

12.

200 µl of NT buffer per 100 µl of DNA are added to the tubes.

13.

 The contents of each tube are transferred to a column, and centrifuged for 1 minute at 11,000g.

14.

 600 µl of an NT3 wash buffer is placed in each column, and centrifuged for 1 minute at 11,000g.

fifteen.

 The columns are centrifuged for 2 minutes at 11,000g.

16.

 51 μl of sterile water is placed on top of the column for 1 minute.

17.

 The column is centrifuged for 1 minute at 11,000g and the labeled labeled DNA is recovered in a tube. Preparation of DNA before hybridization

18.

 50 μl of Cot-1 DNA (Roche) are added to each tube.

19.

 The volume is adjusted to 200 μl using 0.3M sodium acetate (pH 5-8).

twenty.

 300 μl of frozen ethanol are added.

twenty-one.

 The mixture is vortexed and incubated in the dark at 700 ° C for 15 minutes.

22

 The tubes are centrifuged for 15 minutes at 11,000g between 4 and 80C.

2. 3.

 The supernatant is removed using a vacuum pump. Preparation of the BAC on a glass slide The DNA in the form of BAC clones is placed in a dehydrated form on a glass plate and fixed using a

UV light sensitive crosslinking agent (350 mJ). Hybridization of labeled DNA / reference DNA (BAC)

24.

 The residue recovered at point 23 is added to 5 μl of ddH2O.

25.

10 μl for 50 μg / μl of yeast tRNA (Invitrogen, Cat # 15401-011) and the mixture is stirred and heated to 950C for 5 minutes.

26.

 15 µl of hybridization buffer (Ambion Slide Hyb # 3 Buffer) preheated to 700C is added.

27.

 The mixture is vigorously stirred for 30 seconds and centrifuged at 13,000 rpm for 30 seconds.

28.

The mixture is heated to 370C for 30 minutes.

5

10

fifteen

twenty

25

30

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29.

 The glass slide is completely covered with the mixture.

30

A clean Lifterslip coverslip (24 x 32 inches, 61 x 81 cm) (Erie Scientific, Cat # 25X60I-2-4789) is placed above the glass slide in order to cover the microarray.

31.

 20 μl of water are added in order to humidify the joints.

32

 The closed hybridization chamber (Hybridization Chamber, Corning, Cat # 2551) is immersed in a 550C water bath for 16 hours.

Post-hybridization wash

33.

 The glass plate is incubated in a first solution of 0.1% SDS 0.1X SSC, at 500C.

The microarray is successively placed in the following wash solutions (in each solution, for 45 s while stirring and for 45 s without stirring): a) 0.1X SSC, 0.1% SDS; b) 0.1X SSC, 0.1% SDS; c) 0.1X SSC; d) 0.1X SSC;

3. 4.

 The glass slide is immersed at room temperature in a marking box filled with a 0.1X SSC solution, then in another box filled with 100% ethanol (CML, Cat # BVITRI-PPL25L).

35

The glass slides are placed in 50 ml conical tubes and centrifuged for several seconds until dry.

36.

 Glass slides are kept in darkness.

Glass slides are analyzed using a fluorescent scanner (GENEPIX 4000B) with a sensitivity adjusted according to the intensity of the marking signal.

The results are analyzed using the BacMagic + image analysis software (developed by ARRAYGENOMICS) to graphically visualize the profiles in the changing mode of staining agent. For each DNA, a text file is exported by BacMagic + and formatted so that it can be used by the seeGH software. Deletions and gains are recorded based on the visualization of the change symmetry of the staining agent in BacMagic +.

Using seeGH, for each group, the annotations are compiled in order to derive a global picture of the detected anomalies and their frequency. In these figures, which are similar to a karyotype table, the marks represent anomalies (red = loss, green = gain) and their location. The sizes of the marks represent their detection frequency in the patient set.

By analyzing Fig. 2, it is possible to create a typical profile of each tumor group according to their grade and stage, which can then be compared with typical profiles of tumor groups.

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Table 2

Earnings displayed after the analysis of the anomalies detected

image2

Table 3

Deletions displayed after analysis of detected anomalies

image3

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From this summary, it seems that pTaG2 are much less altered than higher grades. Some abnormalities appear to be characteristic of low grade tumors. As described in the literature, deletion 9 is also found here, appearing to be more related to the onset and frequently associated with pTas. In the present case, the 3q and 5p gains also appear to be characteristic of the lower grades (here, it is not a

5 iso5p as described for higher grades, since there is no gain 5q).

Gain anomalies for high grades appear gradually: 8q, 1q, 11q but also 16 and 18, which seems to occur more frequently in stages T2 or higher.

Loss anomalies for high grades are 6q, 11p, and 13, but also 18q, it seems that the last event therefore occurs with delayed tumor progression.

10 IV-CALCULATION AND DEFINITION OF SENSITIVITY AND SPECIFICITY

Sensitivity: ability to detect sick patients.

image4

Specificity: ability of the trial to determine which subjects are healthy.

image5

15 Thanks to the use of tumors known to the inventors, it is possible to compute the sensitivity of the assay. This will allow the ability of the present technique to detect both low grade and high grade tumors that are evaluated. Both groups will be separated in the stones, because low-grade tumors are less difficult to detect. Routine tests, such as urinary cytology, only detect 50% of these cancers in their initial phase.

20 Existing trials are characterized by their sensitivity and specificity as computed in the scientific literature (expressed by a minimum and a maximum since these values vary according to each study - Sources: Biomarkers for detection and surveillance of bladder cancer, Lorne I. Budman , MSc, Wassim Kassouf, MD, and Jordan

R. Steinberg, MD of the Division of Urology, McGill University Health Center, Montreal, Quebec), as indicated in Table 4 below.

25 Table 4

Sensitivity and specificity for existing tests:

image6

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The sensitivity is determined for a given number of markers. For each of these, a sensitivity value can be computed. Obviously, it is not possible for a single genetic marker to result in a reliable solution. However, the combination of 25 markers seems to be considerably more judicious, as shown by the assessments summarized in Tables 5 and 6.

Table 5

Summary evaluation based on the 25 markers

image7

Table 6

Summary evaluation based on the 25 markers

image8

Specificity: 6 negative DNA (DNA without anomalies): 6 / (6 + 0)) * 100 = 100%

The 10 anomalies that have been classified in the low-grade group can be taken as models for tumors in their onset phase, which have not yet become invasive.

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Generally, when the tumor is not invasive but high grade, or when it is already in stage T1 or T2 or higher, the number of abnormalities detected in the markers of this invention is increased.

For high grades, the method confirms the importance of chromosomes 1, 7, 8, 11, 13, 14, 17, and 18.

However, it should be noted that the sensitivity of each of the markers considered separately does not exceed 36% (as is the case for cytology or NMP22 in some studies).

On the other hand, the grouping of all the genetic markers selected by the inventors for the present invention provides very good sensitivity, even for strictly low degrees (the result obtained with the present invention is ONLY as satisfactory as the combination ImmunoCyt / uCyt + and cytology used in certain studies).

10 Similarly, the specificity (computed for normal samples or for DNA extracted from the urine of patients with negative endoscopy and negative follow-up results) reaches 100%.

V-Verification in urine

Urine samples were taken from patients on diagnosis or follow-up.

Routine endoscopy was performed. The patients were grouped as shown in Fig. 2.

15 The method was implemented in the same manner as for frozen tumors (see part II).

The results are analyzed using the BacMagic + software to graphically display the profiles in the staining agent change mode. For each DNA, a text file is exported by BacMagic + and formatted so that it can be used by the seeGH software. Deletions and gains are recorded based on the visualization of the change symmetry of the staining agent in BacMagic +.

20 Using seeGH, for each group, the annotations are compiled in order to derive a global picture of the detected anomalies and their frequency. In these figures, which are similar to a karyotype table, the marks represent anomalies (red = loss, green = gain) and their location. The sizes of the brands represent their detection frequency in the set of patients.

For tumors of the upper urinary system, it is possible to derive a summary evaluation of the abnormalities detected 25 (Tables 7 and 8).

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Table 7

Summary evaluation of the anomalies detected (gains)

image9

Table 8

Summary evaluation of the anomalies detected (deletions)

image10

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For patients with positive endoscopy, the characteristic abnormalities of bladder cancers are recognized, with some variations listed here.

In addition, the anomaly distribution table will be used in an attempt to determine the extent of these detected tumors.

For bladder tumors, this is also done by deriving a summary evaluation of the alterations detected.

Table 9

Summary evaluation of the anomalies detected (gains)

image11

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Table 10

Summary evaluation of the anomalies detected (deletions)

image12

The results listed in Tables 9 and 10 show that many characteristic anomalies are detected in

5 patients with positive endoscopy. However, suspicious endoscopies reveal fewer alterations. In patients for whom the diagnosis by endoscopy was not fully determined, the follow-up showed that these patients had progressed unfavorably. Thus, it seems that a suspicious endoscopy would have been clarified by the inventors' trial before reaching the 6-month follow-up.

It should also be noted that patients with negative endoscopy as well as those for whom follow-up

10 of 12 months did not reveal anything, they showed no positivity for the areas tested. (Only a few clones showed variations, but these were not areas that were intended to be tested, and corresponded to polymorphic variations of clones distributed on the slides. These clones will have to be deleted in the coming versions of this slide, due to their varied nature and position almost centromeric).

If the analysis is carried out on a case-by-case basis in each of the DNA extracted from the urine of the urine of

In a patient with positive endoscopy, the summary table of the present invention, which combines a low grade or a high grade, can be applied.

Specific examples:

DNA 34016: UUTT with positive endoscopy. Deletions 9p and 9q, indicative of low grades were detected. The urologist associated with the inventors performed a biopsy after the test. A grade 1 or 2 was determined.

20 DNA 33276: UUTT with positive endoscopy. Numerous anomalies (8 in total) are detected, including 9q, 11p, 8p and 1q22-q2, which are characteristic of high degrees. After the biopsy, a G3 grade was confirmed.

DNA 32479: Bladder tumor with suspicious endoscopy but positive follow-up at 6 months: only a gain of 19 was detected. This small number of abnormalities and the indeterminate influence of the gain of 19, allows the inventors to associate this tumor with a degree low. Biopsy indicated grade G1 / G2.

25 A biopsy was performed in most of the patients whose urine was tested in this study, provided that their endoscopy was positive. At the time the BCA trial was carried out, the biopsy results were not available.

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This test was thus carried out in blind mode. Their results can be summarized for bladder cancer as shown in Table 11 below.

Table 11

Bladder tumor: positive endoscopy: 8 urine samples

image13

It should be noted that for all patients for whom the result of the biopsy was ambiguous, it would have been possible to predict the grade of the tumor. In addition, in some cases, a more accurate analysis of the altered markers provides information about the aggressive nature of the tumor, its metastatic potential and its possible progression. In addition, some biopsies that were too restricted could not be analyzed, while the present invention

10 is able to provide a clear result regarding the grade of these tumors.

Claims (12)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 1. A method for in vitro detection of bladder cancer in a patient, characterized in that it comprises the stages of:

(i)

extracting the DNA contained in a urine sample taken from said patient;

(ii)

 fragment the DNA extracted in step (i);

(iii) labeling the DNA fragments obtained uniformly with a labeling agent so that a set of labeled DNA is formed;

(iv)

 forming at least one aliquot of the labeled DNA set and contacting each aliquot with a reference DNA set, said contact being made under conditions that allow specific hybridization of the labeled DNA fragments with said reference DNA; said reference DNA comprising the DNA sequences included in each of the following loci present in human chromosomes: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7 , 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22;

(v)

 remove labeled DNA fragments that do not specifically hybridize with reference DNAs;

(saw)

 determine the intensity of the signal produced by the hybridized labeled fragments with each of the selected reference DNAs;

(vii) determine, for each reference DNA, the deviations between the signals obtained in comparison to those obtained with a control DNA of a healthy patient; (viii) derive the patient's cancer stage from said deviations observed for loci 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q , 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22qter, 17p, 19 and 22. 2. A method according to claim 1, characterized in that the reference DNAs consist of human chromosome DNA sequences with a length between 1,000 and 200,000 bp (base pairs), preferably between

2,000

 and 180,000 bp, and even more preferably between 5,000 and 160,000 base pairs.

3.

A method according to claim 2, characterized in that the reference DNAs consist of BAC clones of the human genome.

Four.

A method according to one of claims 1 to 3, characterized in that a first and a second marker are used respectively to label the DNA fragments extracted from urine and the control DNA of a healthy patient.

5.

A method according to claim 4, characterized in that the first and second markers are fluorescent markers that emit at different wavelengths, preferably Cy3 and Cy5.

6.

A method according to any one of claims 1 to 5, characterized in that the degree of the tumor cells in step (viii) is determined as a function of the deviations observed with respect to the reference DNAs.

7.

A method according to any one of claims 1 to 6, characterized in that the reference DNA is deposited separately in a microarray, before hybridization with the labeled DNA fragments.

8.

A method according to any one of claims 1 to 7, characterized in that the DNA fragments taken from the patient in step (iii) are divided into two sets, one being marked with a first marker and the second with a second marker, also dividing the Stage control DNA (vii) in two sets, one being marked with the first marker and the second with the second marker, cross-determining the signal deviation in the stage (vii) between each set so marked.

9.

 A DNA microarray for the detection of bladder cancer, characterized in that its surface consists of between 50 and 1,000 reference DNA deposits, said reference DNA consisting of sequences included in each of the human chromosome loci: 1p, 3q , 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22 -qter, 17p, 19 and 22.

10.

A DNA microarray according to claim 9, characterized in that it comprises between 50 and 400, preferably between 300 and 400 of said reference DNA deposits on its surface.

eleven.

Use of the DNA chip of any of claims 9-11 for in vitro urinary detection of bladder cancer.

twenty-one